The invention provides a method for in-vitro regeneration and proliferation of excised shoot meristems of Citrus aurantifolis (Christm.) Swingle and C. sinensis (L.) Osbeck.
In essence, the present invention discloses sequential culture conditions, including growth media for in vitro culture of excised shoot meristems taken from field-grown mature trees of Citrus aurantifolis (Christm.) Swingle and C. sinensis (L.) Osbeck (sweet orange), their regeneration into individual shoots or proliferation, rooting of the isolated meristem-regenerated shoots, hardening of such in vitro-raised plantlets and their successful ex vitro growth.
Citrus is the most important fruit of the world, second to grape in commerce and the third important fruit of India so far as production is concerned. However, considering the nutritional value for human health and the large number of products made from it, Citrus may qualify to be the number one fruit of the world.
Citrus plant is vulnerable to infection by a number of pathogens, particularly viruses, viroid, mycoplasma and BLO (bacteria-like organism) besides bacteria and fungi (Spiegel-Roy, P. and Goldschmidt, E. F. 1996. Biology of Citrus, Cambridge University Press, U.K.), which take a heavy toll of citrus fruit production. According to the latest figures available, citrus produce (oranges) in India is less than that produced in Spain in 1992-93.
Also, whilst Spain is the largest exporter of citrus fruit, India exports none (Spiegel-Roy, P. and Goldschmitt, E. F. Biology of Citrus, Cambridge University Press, U.K.), which is paradoxical in view of the far greater citrus orchard area in India as compared with Spain.
The enviable position acquired by Spain in citrus production is mainly due to tissue culture application, particularly micrografting, through which virus-free orchards have been produced, since shoot meristem in citrus is free from viruses and hence, the resulting plant from it is also free from viruses (Murashige, T., Bitters, W. P., Rangan, T. S., Nauer, E. M., Roistacher, C. N. and Holliday, P. B. 1972. A technique of shoot apex grafting and its utilization towards recovering virus-free Citrus clones. Hort Science 7: 118-119; Navarro, L., Roistacher, C. N. and Murashige, T. 1975. Improvement of shoot-tip grafting in vitro for virus-free citrus. J. Amer. Soc. Hort. Sci. 100: 471-479; Navarro, L. 1992. Citrus shoot tip grafting in vitro. In: Biotechnology in Agriculture and Forestry, Vol. 18. Ed. Y. P. S. Bajaj, pp.327-338, Springer-Verlag, Berlin).
Shoot meristem alone, i.e., consisting of apical dome or accompanied by 1, 2 or 3 youngest leaf primordia, measuring 1 mm or less than 1 mm in length comprises meristem culture. It is the best explant for cloning as well as for elimination of pathogens, as its constituent cells are genetically uniform and mostly free from pathogens. Culture of shoot meristem of Citrus is extremely significant, as it has been reported to be free from viruses, viroid, BLO, mycoplasma, bacteria and fungi (Bitters, W. P. and Murashige, T. 1967. A place for tissue culture in citrus research. Calif. Citrograph 52: 226, 228, 270-272, 304, 306; Navarro, L., Civerolo, E./L., Juarez, J. and Garnsey, S. M. 1991. Improving therapy methods for citrus germplasm exchange. In: Proc. Eleventh IOCV Conference, IOCV, Riverside, pp.400-408). Thus, it will result in production of clean stocks of citrus propagules unassociated with undesirable juvenile characters found in nucellars.
Efforts to culture shoot meristem of citrus started during mid sixties, but success could not be achieved (Murashige, T., Bitters, W. P., Rangan, T. S., Nauer, E. M., Roistacher, C. N. and Holliday, P. B. 1972. A technique of shoot apex grafting and its utilization towards recovering virus-free Citrus clones. HortScience 7: 118-119). Since then, despite concerted efforts made world over, success continued to elude citrus meristem culture. As an alternative to shoot meristem culture, micrografting as aseptically grafting shoot meristem of scion onto the epicotyl of a rootstock, has been resorted to in citrus, despite being very tedious and time consuming and requiring great skill and dexterity that too with only about 40% success (Murashige, T., Bitters, W. P., Rangan, T. S., Nauer, E. M., Roistacher, C. N. and Holliday, P. B. 1972. A technique of shoot apex grafting and its utilization towards recovering virus-free Citrus clones is described in HortScience 7: 118-119; Navarro, L., Roistacher, C. N. and Murashige, T. 1975). Improvement of shoot tip grafting in vitro for virus-free Citrus. is described in J. Amer. Soc. Hort. Sci. 100: 471-479; Navarro, L. 1992. Citrus shoot tip grafting in vitro is described in Biotechnology in Agriculture and Forestry, Vol. 18. Ed. Y. P. S. Bajaj, pp.327-338, Springer-Verlag, Berlin. Nevertheless, the micrografting experiments established that by employing meristems of citrus, viruses and other pathogens have been eliminated, resulting in increased fruit production Navarro, L., Civerolo, E. L., Juarez, J. and Garnsey, S. M. 1991. Improving therapy methods for citrus germplasm exchange is described in Proc. Eleventh IOCV Conference, IOCV, Riverside, pp.400-408.
The main objective of the present invention is to develop a method for production of viable and fertile Citrus plants through in vitro culture of 0.2 to 0.5-mm-long shoot meristems without intervening callusing.
Another objective is to provide growth media and sequential culture conditions, including the physical state of the substratum for optimum regeneration and proliferation of shoot meristems.
Still, other objective is to provide a method for rapid mass production of true-to-type and pathogen-free propagation stocks of both these important scion species.
Accordingly, the invention provides a method for regenerating viable and fertile Citrus plants culture from explants of field-grown mature trees of Citrus aurantifolia and C. sinensis, said method comprising the steps of:
a) cutting an explant from a Citrus plant selected from Citrus aurantifolia and C. sinensis said explant consisting of single-node stem segments from fresh shoots of field-grown mature trees,
b) decontaminating said explant by removing from its surface any contaminant which are harmful to the tissue culture process,
c) incubating the surface-sterilized explant at a temperature between 27xc2x0 C. to 30xc2x0 C. in the presence of ca.3 klux white fluorescent light for 15 hrs. a day, in agarified medium-A and B respectively for a period ranging between 20-30 days, to develop axillary buds and obtain 70-80% infection-free cultures,
d) subculturing the explant of step (c) along with sprouted axillary buds at least 4 times in medium A and B to produce several aseptic fast-growing shoots,
e) excising the meristem domes along with youngest 2-3 leaf primordia of length between 0.2-0.5 mm from the aseptically grown shoots and culturing said shoots in medium C at a temperature between 27xc2x0 C. and 30xc2x0 C. in the presence of ca.3 klux white fluorescent light for a period of about 15 days to generate shoots of length of ca. 8 mm,
f) subculturing the meristem-regenerated shoots on filter paper bridge employing liquid medium-D in order to obtain healthy shoots of an average length of about 1.5 cm without intervening callusing within a period of 20-25 days,
g) proliferating the meristem-regenerated shoots by repeated subculture in medium-A and medium-B, respectively to obtain an average of about 10 well-developed shoots within 4 subcultures of 30 days each,
h) rooting of the isolated well-developed shoots of C. aurantifolia to the extent of about 100% in 15 days, and of C. sinensis to the extent of about 90% in 25 days, in agarified media-E and F respectively.
i) transferring the freshly developed roots of C. sinensis to medium-G while allowing the shoots of C. aurantifolia to grow in the same medium for a further period of 15 days for development of tap roots,
j) hardening the rooted shoots ex vitro in the liquid medium-H for 10 days in a hardening chamber, wherein the relative humidity is regulated from high (ca. 90%) to gradually low (ca. 60%) under ca. 3 klux light intensity from white fluorescent tubes supplemented with light from incandescent lamps at 26xc2x0 to 28xc2x0 C. and allowing the shoots to grow in the same medium under incubation conditions for 30 days,
k) transferring the hardened shoot meristem-raised plants to soilrite in pro-trays for ex vitro growth at relative humidity of 90% to 60% at a temperature of 27xc2x0 C. to 30xc2x0 C. in the presence of white fluorescent light for a period of about 7 days to ensure 95% to 100% survival of the shoot meristem raised Citrus plants.
In an embodiment, the explant from a Citrus plant comprises shoot meristems of length of about 0.2 to 0.5 mm or single node stem segments.
In another embodiment, the explant is decontaminated by surface-sterilizing the nodal stem segments obtained from C. aurantifolia and C. sinensis, washing the said segments with tap water, followed by pre-treating with about 5% xe2x80x9cLabolenexe2x80x9d solution for 5 minutes, rinsing with distilled water followed by a quick dip in 95% ethanol and surface-sterilizing with 0.1% HgCl2 solution for about 15 minutes and washing the surface-sterilized nodal steam segments thoroughly with sterile distilled water followed by cutting off the affected internodal portions on either side of the node and preparing the explant for inoculation.
The Applicants hereby state that various culture media have been used in the method of for regeneration of citrus plants as described in the foregoing paragraphs.
The various media used in the process of the invention are described hereinbelow and represented in table 1. Accordingly, medium A comprises:
Further medium B comprises:
Medium C comprises:
Medium D comprises:
Medium E comprises:
Medium F comprises:
Medium G comprises:
Medium H comprises:
It would be pertinent to note that the various combination of the media hereinlisted would be apparent to those skilled in the art. Such media are deemed to fall within the scope of the present invention.
The tissue culture method for regenerating viable and fertile Citrus plants from field grown trees of C. aurantifolia and C. sinensis is described in further detail as under:
a) excising the single-node stem segments from fresh growth of shoots wherein the said method of field-grown mature elite trees of C. aurantifolia and C. sinensis during the month of February,
b) surface-sterilizing the nodal stem segments of both the Citrus species by washing the said segments with tap water, followed by pre-treating with about 5% xe2x80x9cLabolenexe2x80x9d, a neutral liquid detergent from Glaxo India Ltd. solution for 5 minutes, then rinsing with single distilled water followed by a quick dip in 95% ethanol and surface-sterilizing with 0.1% HgCl2 solution for about 15 minutes and washing the surface-sterilized nodal steam segments thoroughly with sterile distilled water followed by cutting off the affected internodal portions on either side of the node and preparing the explants for inoculation;
c) incubating the surface-sterilized explants of C. aurantifolia and C. sinensis under ca. 3 klux white fluorescent light for 15 h a day at 27xc2x0xc2x11xc2x0 C. temperature in agarified medium-A and medium-B respectively, for a period ranging between 20-30 days for axillary bud sprouting to obtain 70-80% infection-free cultures of C. aurantifolia and C. sinensis, 
d) subculturing the explants along with sprouted axillary buds for at least 4 times in the same respective media as in step (c) to produce enormous number of aseptically fast-growing shoots,
e) excising the meristem domes along with youngest 2-3 leaf primordia ranging in length between 0.2-0.5 mm from the aseptically grown shoots followed by their culture on the liquid medium-C under the same controlled light and temperature conditions as in step (c) at least for a period of 15 days to let them attain a length of ca. 8 mm,
f) subculturing the meristem-regenerated shoots on filter paper bridge employing liquid medium-D in order to obtain healthy shoots of an average length of about 1.5 cm without intervening callusing within a period of 20-25 days,
g) proliferating the meristem-regenerated 1.5-cm-long shoots of C. aurantifolia and C. sinensis by repeated subculture in medium-A and medium-B, respectively to obtain well-developed shoots to the extent of an average 10 shoots and 6 shoots, respectively, within 4 subcultures of 30 days each,
h) rooting of the isolated well-developed shoots of C. aurantifolia to the extent of 100% in 15 days, and of C. sinensis to the extent of 90% in 25 days, in agarified media-E and F respectively.
i) transferring the just rooted of C. sinensis to medium-G while those of C. aurantifolia were left to grow in the same medium for a further period of 15 days in order to obtain almost a tap root-like system.
j) hardening the rooted shoots ex vitro in the liquid medium-H for 10 days in a hardening chamber, where the R.H. being regulated from high (ca. 900/%) to gradually low (ca. 60%) under ca. 3 klux light intensity from white fluorescent tubes supplemented with light from incandescent lamps at 27xc2x0xc2x11xc2x0 C. and allowing to grow the rooted shoots further in the same medium and incubation conditions for another 30 days.
k) transferring the hardened shoot meristem-raised plants to soilrite in pro-trays for ex vitro growth under the same humidity regime and incubation conditions as stated in step (j) for 7 days to obtain their 95% to 100% survival.
It took ca. 18 months to obtain well-developed healthy plants of an average height of 15 cm in potted soil, after the first establishment in aseptic culture of nodal explants taken from field-grown trees of Citrus.